Systems and methods for growing a biofilm of probiotic bacteria on solid particles for colonization of bacteria in the gut

ABSTRACT

The present invention provides a method, wherein the method forms a biofilm, wherein the biofilm comprises a population of at least one bacterial strain attached to particles, wherein the biofilm is configured to colonize a gut of a subject in need thereof for at least five days, when ingested by the subject, the method comprising: a. obtaining a population comprising at least one strain of bacteria; b. inoculating a growth medium containing particles with the population comprising at least one strain of bacteria; c. incubating the particles with the population comprising at least one bacterial strain for a time sufficient for the population of at least one strain of bacteria to attach to the particles; and d. culturing the population comprising at least one strain of bacteria attached to the particles in a growth medium, for a time sufficient to form a biofilm.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase of PCT Patent Application No.PCT/IB2016/000933 having International filing date of May 9, 2016, whichclaims the benefit of priority to U.S. Provisional Patent ApplicationSer. No. 62/159,846, filed on May 11, 2015, and U.S. Provisional PatentApplication Ser. No. 62/159,849, filed on May 11, 2015, the entirecontents of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to system and method for growing andencapsulating at least one strain of bacteria in a biofilm form,configured for pH dependent targeted release of the bacterial biofilm inthe gastrointestinal tract.

SUMMARY

In one embodiment, the present invention provides a method,

-   -   wherein the method forms a biofilm,    -   wherein the biofilm comprises a population of at least one        bacterial strain attached to particles,    -   wherein the biofilm is configured to colonize a gut of a subject        in need thereof for at least five days, when ingested by the        subject, the method comprising:        -   a. obtaining a population comprising at least one strain of            bacteria;        -   b. inoculating a growth medium containing particles with the            population comprising at least one strain of bacteria;        -   c. incubating the particles with the population comprising            at least one bacterial strain for a time sufficient for the            population of at least one strain of bacteria to attach to            the particles; and        -   d. culturing the population comprising at least one strain            of bacteria attached to the particles in a growth medium,            for a time sufficient to form a biofilm.

In one embodiment, the biofilm comprising a population of at least onebacterial strain attached to particles is encapsulated with a compoundconfigured to release the at least one bacterial strain at a pH found inthe intestine of the animal.

In one embodiment, the compound configured to release the at least onebacterial strain at a pH found in the intestine of the animal isalginate.

In one embodiment, the population of at least one strain of bacteriaattached to the particles is cultured in the growth medium under flowconditions.

In one embodiment, the population of at least one strain of bacteriaattached to the particles is cultured in the growth medium under staticconditions.

In one embodiment, the population of at least one strain of bacteriaattached to the particles is first cultured in the growth medium understatic conditions, followed by culture in the growth medium under flowconditions.

In one embodiment, the population of at least one strain of bacteriaattached to the particles is cultured under anaerobic conditions.

In one embodiment, the population of at least one strain of bacteriaattached to the particles is cultured under aerobic conditions.

In one embodiment, the particles are porous, and selected from the groupconsisting of: seeds, dicalcium phosphate, clay, sand and cellulose.

In one embodiment, the population comprising at least one bacterialstrain is derived from gut microflora.

In one embodiment, the population comprising at least one bacterialstrain is Lactobacillus plantarum.

In one embodiment, the population comprising at least one bacterialstrain is Acetobacter pomorum.

In one embodiment, the biofilm formed by the method is configured for pHdependent targeted release of the bacterial biofilm in thegastrointestinal tract.

In one embodiment, the biofilm comprises two or more strains ofbacteria.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an illustration of an exemplary embodiment of the presentinvention, showing a flow system used according to the methods accordingto some embodiments of the present invention.

FIG. 2A to 2C show images of some exemplary embodiments of biofilmsgenerated by the methods according to some embodiments of the presentinvention.

FIG. 3 shows the acidity tolerance of a biofilm according to someembodiments of the present invention.

FIG. 4 shows the acidity tolerance of another biofilm according to someembodiments of the present invention.

FIG. 5 shows the tolerance of a biofilm according to some embodiments ofthe present invention to lyophilization.

FIG. 6 shows the acidity tolerance of another biofilm according to someembodiments of the present invention.

FIG. 7 shows the ability of a biofilm according to some embodiments ofthe present invention to colonize the gut of an animal model, using theindicated compositions.

FIG. 8 shows the pH-dependent release of bacteria from biofilmsaccording to some embodiments of the present invention.

FIG. 9 shows the ability of another biofilm according to someembodiments of the present invention to colonize the gut of an animalmodel

FIG. 10 shows the ability of another biofilm according to someembodiments of the present invention to colonize the gut of an animalmodel, compared to other biofilms formed using other methods.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following subsectionsthat describe or illustrate certain features, embodiments orapplications of the present invention.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrases “in one embodiment” and “in someembodiments” as used herein do not necessarily refer to the sameembodiment(s), though it may. Furthermore, the phrases “in anotherembodiment” and “in some other embodiments” as used herein do notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or”operator, and is equivalent to the term “and/or,” unless the contextclearly dictates otherwise. The term “based on” is not exclusive andallows for being based on additional factors not described, unless thecontext clearly dictates otherwise. In addition, throughout thespecification, the meaning of “a,” “an,” and “the” include pluralreferences. The meaning of “in” includes “in” and “on.”

In some embodiments, the present invention relates to system and methodfor growing and encapsulating at least one strain of bacteria in abiofilm form, configured for pH dependent targeted release of thebacterial biofilm in the gastrointestinal tract.

In one embodiment, the present invention provides a method,

-   -   wherein the method forms a biofilm,    -   wherein the biofilm comprises a population of at least one        bacterial strain attached to particles,    -   wherein the biofilm is configured to colonize a gut of a subject        in need thereof for at least five days, when ingested by the        subject, the method comprising:        -   a. obtaining a population comprising at least one strain of            bacteria;        -   b. inoculating a growth medium containing particles with the            population comprising at least one strain of bacteria;        -   c. incubating the particles with the population comprising            at least one bacterial strain for a time sufficient for the            population of at least one strain of bacteria to attach to            the particles; and        -   d. culturing the population comprising at least one strain            of bacteria attached to the particles in a growth medium,            for a time sufficient to form a biofilm.

In some embodiments, the time sufficient for the population of at leastone strain of bacteria to attach to the particles is from 2 hours to 12hours. In some embodiments, the time sufficient for the population of atleast one strain of bacteria to attach to the particles is 2 hours. Insome embodiments, the time sufficient for the population of at least onestrain of bacteria to attach to the particles is 4 hours. In someembodiments, the time sufficient for the population of at least onestrain of bacteria to attach to the particles is 6 hours. In someembodiments, the time sufficient for the population of at least onestrain of bacteria to attach to the particles is 8 hours. In someembodiments, the time sufficient for the population of at least onestrain of bacteria to attach to the particles is 10 hours. In someembodiments, the time sufficient for the population of at least onestrain of bacteria to attach to the particles is 12 hours.

In some embodiments, the time sufficient to form a biofilm is from 12hours to 48 hours. In some embodiments, the time sufficient to form abiofilm is 12 hours. In some embodiments, the time sufficient to form abiofilm is 14 hours. In some embodiments, the time sufficient to form abiofilm is 16 hours. In some embodiments, the time sufficient to form abiofilm is 18 hours. In some embodiments, the time sufficient to form abiofilm is 20 hours. In some embodiments, the time sufficient to form abiofilm is 22 hours. In some embodiments, the time sufficient to form abiofilm is 24 hours. In some embodiments, the time sufficient to form abiofilm is 26 hours. In some embodiments, the time sufficient to form abiofilm is 28 hours. In some embodiments, the time sufficient to form abiofilm is 30 hours. In some embodiments, the time sufficient to form abiofilm is 32 hours. In some embodiments, the time sufficient to form abiofilm is 34 hours. In some embodiments, the time sufficient to form abiofilm is 36 hours. In some embodiments, the time sufficient to form abiofilm is 38 hours. In some embodiments, the time sufficient to form abiofilm is 40 hours. In some embodiments, the time sufficient to form abiofilm is 42 hours. In some embodiments, the time sufficient to form abiofilm is 44 hours. In some embodiments, the time sufficient to form abiofilm is 46 hours. In some embodiments, the time sufficient to form abiofilm is 48 hours.

In some embodiments, the population of at least one strain of bacteriaattached to the particles is cultured in the growth medium under flowconditions. As used herein, the term “flow conditions” refers to themovement of culture medium in relation to bacteria attached to asurface, wherein the movement of the culture medium exerts a shear forceon the bacteria.

Without intending to be limited to any particular theory, culturing thepopulation of at least one strain of bacteria attached to the particlesunder flow conditions creates even gentle shear forces on the growingbiofilm and increases the fast creation of the biofilm (e.g., in ashorter period of time compared with typical stationary growth methods).In some embodiments, a flowing system allows for the introduction offresh culture medium to the growing biofilm, and the removal ofbacterial waste.

In some embodiments, the flow conditions comprise a flow rate of 10ml/hour to 100 ml/hour. In some embodiments, the flow conditionscomprise a flow rate of 20 ml/hour. In some embodiments, the flowconditions comprise a flow rate of 30 ml/hour. In some embodiments, theflow conditions comprise a flow rate of 40 ml/hour. In some embodiments,the flow conditions comprise a flow rate of 50 ml/hour. In someembodiments, the flow conditions comprise a flow rate of 60 ml/hour. Insome embodiments, the flow conditions comprise a flow rate of 70ml/hour. In some embodiments, the flow conditions comprise a flow rateof 80 ml/hour. In some embodiments, the flow conditions comprise a flowrate of 90 ml/hour. In some embodiments, the flow conditions comprise aflow rate of 100 ml/hour. In some embodiments, the flow conditionscomprise a flow rate of 10 ml/hour.

In some embodiments, the flow conditions comprise shaking the culture ofbacteria from 90 to 150 rpm. In some embodiments, the flow conditionscomprise shaking the culture of bacteria at 100 rpm. In someembodiments, the flow conditions comprise shaking the culture ofbacteria at 110 rpm. In some embodiments, the flow conditions compriseshaking the culture of bacteria at 120 rpm. In some embodiments, theflow conditions comprise shaking the culture of bacteria at 130 rpm. Insome embodiments, the flow conditions comprise shaking the culture ofbacteria at 140 rpm. In some embodiments, the flow conditions compriseshaking the culture of bacteria at 150 rpm.

In some embodiments, culturing the population of at least one strain ofbacteria attached to the particles under flow conditions results inproducing a robust and healthy biofilm in a shorter period of timecompared with typical methods (e.g., but not limited to, 5, 10, 20, 25,50% less time). In some embodiments, the resulting biofilm has anincreased resilience to harsh conditions when compared with otherculturing methods, and is further detailed herein.

Referring to FIG. 1, an illustration of an exemplary embodiment of thepresent invention, showing a flow system according to some embodimentsof the present invention is shown. Referring to FIG. 1, the systemincludes a container that contains the solid particles for biofilmcultivation, a source of growth medium, tubes that conduct the growthmedium in and out of the container, and a pump that moves the mediumthrough the tubes. The fluid outlet from the container can return to themedium reservoir for recycling, or can be drained away. In someembodiments, this flow system can be closed, open, or semi-closed. Theclockwise moving arrows in FIG. 1 represent the direction of the flow,and are for illustration purposes only.

In some embodiments, the population of at least one strain of bacteriaattached to the particles is cultured in the growth medium under staticconditions. As used herein, the term “static conditions” refers toculture conditions where no shear forces exerted on the bacteria.

In some embodiments, the population of at least one strain of bacteriaattached to the particles is first cultured in the growth medium understatic conditions, followed by culture in the growth medium under flowconditions.

In some embodiments, the population of at least one strain of bacteriaattached to the particles is cultured under anaerobic conditions. Asused herein, the term “anaerobic conditions” refers to cultureconditions comprising the absence of free or bound oxygen.

In some embodiments, the population of at least one strain of bacteriaattached to the particles is cultured under aerobic conditions. As usedherein, the term “aerobic conditions” refers to culture conditionscomprising the presence of free or bound oxygen.

Particles

In some embodiments, the particles are porous, and selected from thegroup consisting of: seeds, dicalcium phosphate, clay, sand, andcellulose.

In some embodiments, the seeds are selected from the group consistingof: pomegranate seeds, and passion fruit seeds. In some embodiments, theseeds are crushed.

In some embodiments, the cellulose particles comprise cellulose soldunder the tradename AVICEL®. In some embodiments, the celluloseparticles comprise cellulose sold under the tradename SOLKA®.

In some embodiments, a plurality of particles is used in the method toform a biofilm according to some embodiments of the present invention.In some embodiments, the particles range from 5 microns to 1 cm indiameter. In some embodiments, the particles are 5 microns in diameter.In some embodiments, the particles are 10 microns in diameter. In someembodiments, the particles are 15 microns in diameter. In someembodiments, the particles are 20 microns in diameter. In someembodiments, the particles are 30 microns in diameter. In someembodiments, the particles are 40 microns in diameter. In someembodiments, the particles are 50 microns in diameter. In someembodiments, the particles are 60 microns in diameter. In someembodiments, the particles are 70 microns in diameter. In someembodiments, the particles are 80 microns in diameter. In someembodiments, the particles are 90 microns in diameter. In someembodiments, the particles are 100 microns in diameter. In someembodiments, the particles are 200 microns in diameter. In someembodiments, the particles are 300 microns in diameter. In someembodiments, the particles are 400 microns in diameter. In someembodiments, the particles are 500 microns in diameter. In someembodiments, the particles are 600 microns in diameter. In someembodiments, the particles are 700 microns in diameter. In someembodiments, the particles are 800 microns in diameter. In someembodiments, the particles are 900 microns in diameter. In someembodiments, the particles are 1 cm in diameter.

Bacterial Strains

In some embodiments, the population comprising at least one bacterialstrain is derived from intestinal flora.

In some embodiments, the population comprising at least one bacterialstrain is a probiotic strain. As used herein, the term “probiotic”refers to a bacterial strain that stimulates the growth ofmicroorganisms, especially those with beneficial properties (such asthose of the intestinal flora).

In some embodiments, the population comprising at least one bacterialstrain is Lactobacillus plantarum.

In some embodiments, the population comprising at least one bacterialstrain is Acetobacter pomorum.

In some embodiments, the biofilm formed by the method is configured forpH dependent targeted release of the bacterial biofilm in thegastrointestinal tract.

In some embodiments, the biofilm comprises two or more strains ofbacteria.

FIG. 2A shows images of some exemplary embodiments of biofilms generatedby the methods of the present invention. In some embodiments, severaltypes of solid particles appropriate for growing probiotic bacteria havebeen tested and the results are shown herein. FIG. 2A shows electronmicroscope images of Lactobacillus plantarum biofilm grown on differentsolid particles, such as, for example, passion fruit seeds, pomegranatecrushed seeds, bentonite clay, sand particles, white clay, SOLKA fibers,dicalcium phosphate (DCP), AVICEL. With the exception of white clay,bacteria grow on all particle types.

FIG. 2B shows images of some exemplary embodiments of biofilms generatedby the methods of the present invention, showing the biofilm growth ofAcetobacter pomorum on passion fruit crushed seeds, pomegranate crushedseeds, and sand. Acetobacter pomorum grew and formed a biofilm onpomegranate seeds. However, very little growth on sand was observed.Acetobacter pomorum did not grow on passion fruit seeds. Other bacteriaspecies tested, for example Pseudomonas spp, did not grow on the solidparticles tested, as shown in FIG. 2C.

Without being bound by theory, different particles providedistinguishable microenvironments for the bacteria to grow on, such aspore size, roughness of the surface, nutrients available in theparticles, viscosity, surface charge, etc., which may influence theability of various bacteria to attach and grow on various kinds ofparticles.

In some embodiments of the methods of the present invention, the method

generates a biofilm containing at least two strains of probioticbacteria (e.g., but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.),where the biofilm is generated using a combination of at least twodifferent particles (e.g., but not limited to, passion fruit seeds,pomegranate crushed seeds, etc.). In some embodiments, for creating suchcombinations, the growth conditions (and, e.g., but not limited to typesof particle(s)) are selected according to the strain(s) for use ingenerating a biofilm. In an exemplary embodiment, if two bacterialstrains will eventually be combined to generate a biofilm, each of thebacterial strains will be grown using the particle best suited for thegrowth of each strain. In some embodiments, when two or more bacterialstrains are grown separately, the bacterial strains are combined duringthe encapsulation process.

Treatment

In some embodiments, a biofilm is administered to an animal in needthereof, to colonize the gut of the animal with the biofilm.

In some embodiments, the biofilm comprising a population of at least onebacterial strain attached to particles is encapsulated with a compoundconfigured to release the at least one bacterial strain at a pH found inthe intestine of the animal.

In some embodiments, the compound configured to release the at least onebacterial strain at a pH found in the intestine of the animal isalginate.

In some embodiments, the pH found in the intestine of the animal is pH8.

In some embodiments, the biofilm is administered to an animal in needthereof in an amount sufficient to colonize the gut. In someembodiments, colonization is confirmed by the presence of the at leastone population of bacteria being present in the feces of the animal forat least 5 days post administration.

In some embodiments, the colonized bacteria derived from the biofilm caninhabit the gut of a mammal for at least one week (e.g., but not limitedto, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. weeks). In some embodiments, thecolonized bacteria derived from the biofilm are sustainable within amammalian gut, i.e., do not die off after 3 days.

In some embodiments, the amount sufficient to colonize the gut is 2×10⁴to 2×10⁹ bacteria per day, for 1 to 5 days. In some embodiments, theamount sufficient to colonize the gut is 2×10⁴ to 2×10⁶ bacteria perday, for 1 to 5 days.

In some embodiments, the amount sufficient to colonize the gut is 2×10⁴bacteria per day, for 5 days. In some embodiments, the amount sufficientto colonize the gut is 2×10⁵ bacteria per day, for 5 days. In someembodiments, the amount sufficient to colonize the gut is 2×10⁶ bacteriaper day, for 5 days. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁷ bacteria per day, for 5 days. In someembodiments, the amount sufficient to colonize the gut is 2×10⁸ bacteriaper day, for 5 days. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁹ bacteria per day, for 5 days.

In some embodiments, the amount sufficient to colonize the gut is 2×10⁴bacteria per day, for 4 days. In some embodiments, the amount sufficientto colonize the gut is 2×10⁵ bacteria per day, for 4 days. In someembodiments, the amount sufficient to colonize the gut is 2×10⁶ bacteriaper day, for 4 days. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁷ bacteria per day, for 4 days. In someembodiments, the amount sufficient to colonize the gut is 2×10⁸ bacteriaper day, for 4 days. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁹ bacteria per day, for 4 days.

In some embodiments, the amount sufficient to colonize the gut is 2×10⁴bacteria per day, for 3 days. In some embodiments, the amount sufficientto colonize the gut is 2×10⁵ bacteria per day, for 3 days. In someembodiments, the amount sufficient to colonize the gut is 2×10⁶ bacteriaper day, for 3 days. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁷ bacteria per day, for 3 days. In someembodiments, the amount sufficient to colonize the gut is 2×10⁸ bacteriaper day, for 3 days. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁹ bacteria per day, for 3 days.

In some embodiments, the amount sufficient to colonize the gut is 2×10⁴bacteria per day, for 2 days. In some embodiments, the amount sufficientto colonize the gut is 2×10⁵ bacteria per day, for 2 days. In someembodiments, the amount sufficient to colonize the gut is 2×10⁶ bacteriaper day, for 2 days. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁷ bacteria per day, for 2 days. In someembodiments, the amount sufficient to colonize the gut is 2×10⁸ bacteriaper day, for 2 days. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁹ bacteria per day, for 2 days.

In some embodiments, the amount sufficient to colonize the gut is 2×10⁴bacteria per day, for 1 day. In some embodiments, the amount sufficientto colonize the gut is 2×10⁵ bacteria per day, for 1 day. In someembodiments, the amount sufficient to colonize the gut is 2×10⁶ bacteriaper day, for 1 day. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁷ bacteria per day, for 1 day. In someembodiments, the amount sufficient to colonize the gut is 2×10⁸ bacteriaper day, for 1 day. In some embodiments, the amount sufficient tocolonize the gut is 2×10⁹ bacteria per day, for 1 day.

In some embodiments, the amount sufficient is administered on a singleparticle. Alternatively, the amount sufficient is administered on aplurality of particles.

In some embodiments, the amount sufficient is mixed with food, andingested.

In some embodiments, the biofilm is administered to the animalimmediately after the biofilm is cultured. Alternatively, the biofilmmay be stored, prior to administration. The biofilm may be storedfrozen, or, alternatively, in a lyophilized form.

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in anon-limiting fashion.

EXAMPLES Example 1: Acidity Tolerance of a Biofilm According to SomeEmbodiments of the Present Invention

The resilience of the biofilm grown on solid particles in the flowingsystem as described above was tested. Specifically, the first parametertested was acidity tolerance, and the results are shown in FIG. 3.

An overnight culture of L. plantarum was inoculated in pomegranatematrix soaked in 25% MRS medium (to encourage biofilm formation,starvation conditions (i.e. less than a 100% concentration of growthmedium) were used) in the matrix container and left still for 2.5 hours(i.e., no mixing), and then the flow system was initiated. Medium wasmoved from the medium reservoir to the matrix container using aperistaltic pump at a speed of 12 ml/hour for a duration of 5 days. Themedium was not recycled. Fresh medium entered the culture and the outletdrained away the used media. As a control, bacteria grown planktonically(i.e., not attached to a particle) were used, resulting in a lack ofbiofilm forming. For the planktonic control, 4-5 colonies of L.plantarum were inoculated in 6 ml of 100% MRS broth and left still inincubator at 37° C. overnight.

To test acidity tolerance, a series of vials with PBS adjusted toincreasing pH using HCl (stock solution 0.5M) prepared in advance tocreate pH 1, 2, 3, and 2 grams of the particles having a biofilm wastransferred into the vials and incubated for 1 hr. The bacteria werethen washed in PBS and 5 microliters were plated as shown in FIG. 3.

For the planktonic control, 100 μl from the overnight culture were takeninto the pH vials and incubated for 1 hr, the bacteria were then washedin PBS and 5 microliters were plated as shown in FIG. 3. As shown inFIG. 3, the biofilm bacteria show a greater resilience to acidity asthey survived well pH 2, while the planktonic bacteria did not survive.

Without intending to be limited to any particular theory, the pH in thestomach is about pH 2. Thus, upon administration of the biofilm to asubject, the biofilm will survive the subject's stomach environment(i.e., pH of 2) and colonize the subject.

Example 2: Acidity Tolerance of Another Biofilm According to SomeEmbodiments of the Present Invention

A second set of experiments were conducted and demonstrate bacterialresilience to acidity (i.e., in the form of a biofilm) is described inFIG. 4 and shown in Tables 1 and 2 below.

An overnight culture of L. plantarum was inoculated into 7 grams ofpomegranate (POM) seed particles soaked in 25% MRS medium (starvationconditions), and left still for 2.5 hours. Next, the flowing system wasconducted for 5 days, using a peristaltic pump that moved the medium atmax speed (about 380 ml/hour). In this experiment, the medium wasrecycled, to compare to the non flowing/stationary control. Forplanktonic control, 4 colonies of L. plantarum were inoculated in 6 mlof 100% MRS broth and left in incubator 37° C. overnight+5 hours.Results are shown in Tables 1 and 2.

TABLE 1 Cell Growth Planktonic Biofilm flow pH (cells/ml) (cells/1 grmatrix) 1.5  2*10⁴ 2.3*10⁴ — — 2 5.6*10⁵ 3.3*10⁵  5*10⁸  4*10⁸ 2.5 3*10⁶ 6.6*10⁶ 4.5*10⁸ 5.6*10⁸ 3 6.3*10⁸ 6.3*10⁸ 4.3*10⁸ 5.1*10⁸ 46.3*10⁸  4*10⁸ 10⁹ 1.3*10⁹ 7.4  1.l*10⁹ 1.1*10⁹  3*10⁸ 3.8*10⁸

TABLE 2 Data of FIG. 4 (Log of Results) Planktonic Biofilm flow pH(cells/ml) (cells/1 gr matrix) 1.5 4.3 4.36 — — 2 5.7 5.51 8.69 8.6 2.56.47 6.81 8.65 8.74 3 8.79 8.79 8.63 8.7 4 8.79 8.6 9 9.11 7.4 9.04 9.048.47 8.57

As shown in FIG. 4, the planktonic bacteria showed decreasing survivalwhen exposed to decreasing pH.

Example 3: Reconstitution of a Biofilm According to Some Embodiments ofthe Present Invention

The biofilm grown and entrapped in alginate recovered after drying. L.plantarum was grown in 25 ml 100% MRS+2 gr POM for 4 days at roomtemperature to form biofilms. For planktonic control, L. plantarum wasgrown in 25 ml 100% MRS for 4 days at room temp. One sample ofPOM+biofilm and control were plated on MRS plates in serial dilutions.Another sample of each was centrifuged briefly, resuspended in 5 mlfreeze drying buffer, lyophilized for 24 hours, and suspended in 25 mLMRS. At this stage (after lyophylization but before further growth),samples were plated by serial dilutions. The remainder of the sample wasleft to grow for 48 additional hours, and plated again. As shown in FIG.5, bacteria that grew as biofilm on solid particles showed an enhancedresistance and survival to lyophilization (i.e., reconstitution afterdrying).

Example 4: Acid Tolerance of E. coli DH5a Cells

Acid tolerance of E. coli strain DH5a was tested under three conditions:

-   -   1. Planktonic—E. coli grown in 20 ml LB starter in shaker for 5        days at 23° C.    -   2. Biofilm static—E. coli grown in 20 ml LB with 2 gr of        different matrixes grown for 5 days in static conditions at 37°        C.    -   3. Biofilm flow—E. coli grown in column flow system with DCP for        5 days at room temperature at a speed of approximately 12        ml/hour fresh 25% LB. Samples were taken from the top of the        column (close to the air surface) and from the bottom of the        column.

E. coli strain DH5a starter was grown overnight in 37° C. shaking. 1041from the starter was transferred to:

-   -   1. 2 gr avicel+20 ml LB—for biofilm static culture.    -   2. 2 gr Solka fibers+20 ml LB—for biofilm static culture.    -   3. 3 gr DCP+20 ml LB—for biofilm static culture.    -   4. 20 ml LB—for planktonic culture.

2 ml of the overnight starter was transferred to 20 ml of LB andinoculated in the column of the flow system. Flow was arrested for 2hours to let the E. coli attach to the DCP. After 2 hours, flow wasturned on for 5 days at room temperature.

After 5 days of incubation, a sample from each of the matrixes from thestatic experiments (DCP, avicel and solka) and a sample from the DCPform the top of the column and DCP form the bottom of the column wastaken and inserted into five different Eppendorf tubes (“Eppendorfs”).The samples were gently washed once with PBS.

From each sample, the following amount of matrix was taken into twovials:

-   -   1. Static DCP—0.02 gr    -   2. Static avicel—0.03 gr    -   3. Static Solka—0.02 gr    -   4. Flow DPC top—0.03 gr    -   5. Flow DCP bottom—0.03 gr

The content of each Eppendorf was gently washed once with PBSX1. Foreach pair of eppendorfs (from each sample), 1 ml of PBSX1 (pH=7.4) or 1ml of PBS (pH=2) was added. The eppendorfs were incubated on their sidefor 1 hour at room temp. The eppendorfs were then centrifuged at 13,000rpm for 2 min, the supernatant was discarded. 1 ml of PBSX1 was added toeach Eppendorf and the eppendorfs were vortexed at full power for 30 secto free the bacteria from the matrix.

Handling of the planktonic culture: 1 ml of culture was transferred toan Eppendorf and centrifuged at full speed for 2 min. Supernatant wasdiscarded and 1 ml of PBS was added. 1041 from this was added to:

-   -   1. Eppendorf with 1 ml of PBSX1 (pH=7.4)    -   2. Eppendorf with 1 ml of PBS (pH=2)

The eppendorfs were incubated for 1 hour at room temp on their side, andthis incubation was followed by centrifugation at 13,000 rpm for 2 min.100 μl of PBSX1 was added to each Eppendorf.

Viable Counts:

10 μl from each eppendorf was transferred to 90 μl of PBSX1. Seven 1:10serial dilutions were conducted. 3 μl from each dilution were plated onan LB plate and left in the incubator overnight.

The calculations were as follows to retain bacteria/ml for planktonic orbacteria/gr:

For matrices: Number of colonies×10^(dilution number)×333.33×(1/grtaken)

For planktonic: Number of colonies×10^(dilution number)×333.3

All materials in this experiment were autoclaved for sterilization. Theresults as shown in the tables below and in FIG. 6 demonstrate that thebiofilm increased the acid tolerance of the bacteria, in the staticconditions and exhibited a greater increase in the flow conditions.

TABLE 3 Results (E. coli DH5α): % survival pH = 2 pH = 7 in pH 2 Topflow biofilm DCP 1.2*10¹⁰ bacteria/gr 2.7*10¹⁰ bacteria/gr 44.4% Bottomflow biofilm DCP 6.6*10⁹ bacteria/gr 1.4*10¹⁰ bacteria/gr 47.1% Staticbiofilm DCP 1.8*10⁹ bacteria/gr 3.8*10⁹ bacteria/gr 47.36%  Staticbiofilm Solka 2.3*10⁸ bacteria/gr 1.8*10⁹ bacteria/gr 12.7% Staticbiofilm Avicel 2.3*10⁸ bacteria/gr 2.6*10⁹ bacteria/gr  8.8% Planktonic0 bacteria/ml 1.9*10⁹ bacteria/ml   0%

TABLE 4 Log Scale % survival pH = 2 pH = 7 in pH 2 Top flow biofilm DCP10.07 10.43 44.4% Bottom flow biofilm DCP 9.8 10.14 47.1% Static biofilmDCP 9.25 9.57 47.36%  Static biofilm Solka 8.36 9.25 12.7% Staticbiofilm Avicel 8.36 9.41  8.8% Planktonic 0 9.27   0%

Example 5: Colonization of Murine Gut Using a Composition According toSome Embodiments of the Present Invention

To test whether the bacteria grown as biofilm show enhanced ability tocolonize the gut, biofilm was prepared as described above, and thebiofilm fed to nude mice. The presence of bacteria in the mouse feceswas tested. The results are shown in FIG. 7.

Protocol:

-   -   1. 6 germ-free mice    -   2. Lactobacillus plantarum biofilm grown on matrix.    -   3. Ground food for mice mixed with sterile matrix only (Grind        together).    -   4. Ground food for mice mixed with L. plantarum biofilm on        matrix (Grind together).    -   5. Ground food for mice mixed with L. plantarum biofilm in        alginate beads.    -   6. Live-dead staining kit (to determine presence of live        bacteria).    -   7. Autoclave matrix for control mice.    -   8. The mice were divided into 3 groups (2 mice for each group):        -   a. Control—mice that are fed food+matrix only.        -   b. Biofilm—mice that are fed with food+biofilm on matrix.        -   c. Biofilm alginate—mice that are fed with food+biofilm on            matrix in alginate beads.    -   9. Feed mice with corresponding food for 7 days (D1-D7).    -   10. Sample stool at day 8 (D8), day 9 (D9), day 10 (D10), Day 11        (D11), day 12 (D12), day 13 (D13) and day 14 (D14).    -   11. Check presence of L. plantarum in stool samples.    -   12. Take section from inner intestine to image biofilm of L.        Plantation on intestine, and stain for live-dead using a        designated staining kit.

FIG. 7 shows that bacteria colonized the gut of the mice. The gut of themice was colonized with the bacteria derived from the biofilm for morethan 3 days, and, in fact, for up to, but not limited to, 14 days.

Example 6: pH-Dependent Release of Bacteria from a Composition Accordingto Some Embodiments of the Present Invention

A biofilm comprising E. coli on DCP was encapsulated in alginate bymixing the DCP with the biofilm in 4% alginate and dropping dropletswith the material into 2% CaCl₂ solution, and a biofilm comprising L.plantarum was grown on top of the alginate beads. The resultingcompositions were then treated according to Example 1 above. The resultsare shown in FIG. 8.

Only Lactobacillus plantarum was released when the beads were insertedinto a solution at pH=2. At pH=8 both E. coli and L. plantarum werereleased from the beads.

Example 7: Colonization of Murine Gut Using a Composition Comprising C.minuta According to Some Embodiments of the Present Invention

A biofilm comprising C. minuta on pomegranate seeds was given to SPFmice once at a concentration of 2*10⁷ bacteria at day 1 of theexperiment mixed with the food. Feces were checked at day 1 (beforeprobiotic treatment), 2, 4, 7, 11 and 15 for the % of C. minuta in thefeces.

The animals were treated as follows (three mice per treatment group):

-   -   1. Control—Food only (6 gr).    -   2. Control—Food (3 gr) mixed with pomegranate grains (3 gr) only    -   3. Experiment—Food (3 gr) mixed with C. minuta biofilm on        pomegranate grains (3 gr).

SPF mice were administered 2*10⁷ C. minuta cells in biofilm onpomegranate grains (C. minuta), pomegranate grains (POM only) or justdiet (HFD) on the first day of the experiment. Feces samples were takenbefore the probiotic treatment (Day 1), 2 days, 4 days, 7 days, 11 daysand 15 days following the probiotic treatment and sent to 16Ssequencing. The percent of C. minuta in the overall population ofbacteria in each feces sample was calculated. The results are shown inFIG. 9. These data show that the biofilm comprising C. minuta wascapable of colonizing the gut of mice for up to 15 days, when theexperiment was terminated, as evidenced by the presence of C. minuta infecal samples.

Example 8: Colonization of Murine Gut Using a Composition According toSome Embodiments of the Present Invention—Comparison with Other Methods

A biofilm comprising L. plantarum was grown according to the conditionsoutline below, and given to SPF mice once at a concentration of 2*10⁹bacteria per day, for 5 days. After this time, mice were fed with foodalone for a further 5 days.

The animals were treated as follows (two mice per treatment group):

-   -   1. Control—Food only    -   2. Control—Food with particles (pomegranate grains POM) only.    -   3. Planktonic. L. plantarum was gown overnight in shaking at 37        C in MRS broth.    -   4. Biofilm static on Pomegranate grains (POM)—5 gr of particles        with bacteria (approximately 10⁶ bacteria/day) (according to the        methods described in DE202013103204)    -   5. Biofilm flow on Pomegranate grains (POM)—1.5 gr of particles        with bacteria.    -   6. Biofilm flow on Pomegranate grains (POM) and lyophilized—1.5        gr of particles with bacteria (according to the methods        described in Biomacromolecules 2013, 14, 3214-3222).    -   7. Commercial probiotic supplement—2 pills per day for 5 day

The amount of Lactobacillus was quantified using colony counts of mice'sfeces taken at Day 5 (the last day of sample admission), Day 3 and Day 5(3 and 5 days following cessation of sample admission). The results areshown in FIG. 10. These data show that the biofilm comprising L.plantarm formed according to the methods of the present invention wascapable of colonizing the gut of mice for up to 5 days, when theexperiment was terminated, as evidenced by the presence of L. plantarumin fecal samples. However, compositions formed according to the methodsdescribed in DE202013103204, or Biomacromolecules 2013, 14, 3214-3222were not capable of colonizing the gut of mice for up to 5 days.

Publications cited throughout this document are hereby incorporated byreference in their entirety. Although the various aspects of theinvention have been illustrated above by reference to examples andpreferred embodiments, it will be appreciated that the scope of theinvention is defined not by the foregoing description but by thefollowing claims properly construed under principles of patent law.

What is claimed is:
 1. A method comprising: providing a bacterialbiofilm comprising at least one bacterial strain attached to particles,obtained by: a. inoculation of a population comprising at least onestrain of bacteria in a growth medium; b. incubation of the particleswith the population comprising at least one bacterial strain for a timesufficient for the population of at least one strain of bacteria toattach to the particles; c. culturing of the population comprising atleast one strain of bacteria attached to the particles in a growthmedium, for a time sufficient to form a bacterial biofilm attached tothe particles, wherein the incubation step occurs in the growth medium,wherein the growth medium exerts a shear force on the bacteria duringthe culturing step, wherein the culturing step comprises culturing underanaerobic conditions, thereby providing the bacterial biofilm comprisingat least one bacterial strain attached to particles; and d.administering the bacterial biofilm attached to particles to a subjectin need thereof, wherein the bacterial biofilm comprising at least onebacterial strain attached to particles is configured to colonize thegastrointestinal tract of a subject, thereby colonizing thegastrointestinal tract of the subject with said bacterial biofilmattached to particles.
 2. The method of claim 1, wherein the populationof at least one strain of bacteria attached to the particles is firstcultured in the growth medium under static conditions, followed byculture in the growth medium under flow conditions.
 3. The method ofclaim 1, wherein the particles are porous particles ranging from 30 to500 microns in diameter.
 4. The method of claim 1, wherein the particlesare selected from the group consisting of: seeds, dicalcium phosphate,and cellulose.
 5. The method of claim 1, wherein the populationcomprising at least one bacterial strain is selected from gutmicroflora.
 6. The method of claim 1, wherein said bacterial biofilm isacid tolerant.
 7. The method of claim 1, wherein said biofilm isconfigured for pH dependent targeted release of the bacterial biofilm inthe gastrointestinal tract.
 8. The method of claim 1, wherein thebiofilm is encapsulated with a compound configured to release the atleast one bacterial strain at a pH found in the intestine of an animal.9. The method of claim 8, wherein said compound is alginate.
 10. Themethod of claim 1, wherein said particle consists of dicalciumphosphate.
 11. The method of claim 1, wherein the at least one bacterialstrain is selected from the group consisting of: Lactobacillus,Christensenella, and Acetobacter.
 12. The method of claim 1, whereinsaid bacterial biofilm comprising a population of at least one bacterialstrain attached to particles is in a lyophilized form.
 13. The method ofclaim 1, wherein said at least one bacterial strain is a probioticstrain.
 14. The method of claim 1, wherein the bacterial biofilm isconfigured to colonize the gut of the subject in need thereof for atleast five days when ingested by the subject.
 15. The method of claim 1,wherein an amount of 2×10⁴ to 2×10⁹ bacteria per day of said bacterialbiofilm is configured to colonize the gut for 1 to 5 days.